The ultimate goal of this SBIR project is to develop a novel proteomic system that will enable the real- time expression profiling of proteins in live murine embryonic stem (mES) cells and in differentiated cells derived there from. The first step involves the establishment of a library of fluorescent trap mES cell lines, each of which will harbor a single fluorescently-tagged protein. The fluorescence in each cell of a particular clonal line will serve as a reporter for the expression and subcellular localization of the tagged protein in that cell line. Such a library of clonal fluorescently-tagged lines can be subjected to quantitative fluorescence microscopy in multiwell live-cell arrays, to enable the simultaneous real-time expression profiling of multiple proteins in live cells under various culture conditions. In Phase I we demonstrated the feasibility of establishing a large and diverse protein-trap library using our approach. This proposed Phase II effort will be focused generating a library of at 1500 unique fluorescent protein-trap mES cell lines. Individual cell lines from such a library of fluorescently protein-trap mES lines can also be used as versatile protein-specific research tools as they can be manipulated in culture into different tissue-types or used for the generation of transgenic mice when desired. Many also these cell lines can also be used to design novel powerful cell-based assays. Therefore this proposed Phase II project will result in the generation of a versatile and diverse proteomic research resource in mES cells. PUBLIC HEALTH RELEVANCE: The ultimate aim of this SBIR project is to develop a system that will enable researchers to monitor the location and levels of hundreds of proteins simultaneously in living cells. Such a system will be useful in researching a wide variety of biological processes, including understanding the molecular basis of a number of human diseases. Further, such a system can be applied to the identification therapeutic targets and the creation of powerful drug-screening platforms to identify potential therapeutic agents that may cure these diseases.